Volume 20, Issue 3, Pages 357-366 (November 2005) Holoenzyme Switching and Stochastic Release of Sigma Factors from RNA Polymerase In Vivo  Marni Raffaelle, Elenita I. Kanin, Jennifer Vogt, Richard R. Burgess, Aseem Z. Ansari  Molecular Cell  Volume 20, Issue 3, Pages 357-366 (November 2005) DOI: 10.1016/j.molcel.2005.10.011 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 The Organization and Sequence of rrn Operons (A) Schematic of the rrn operons. (B) Sequence of the rrnB P1 promoter region from −60 to +10. The transcription initiation site (+1) is indicated. −10 and –35 consensus regions recognized by Eσ70 are underlined. Matches to consensus sequences for EσS and Eσ32 are indicated above and below, respectively. Consensus sequences for EσS are from Gaal et al. (2001) (top) and Weber et al. (2005) (bottom). Consensus sequences for Eσ32 are from Wang and deHaseth (2003) (top) and Cowing et al. (1985) (bottom). Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Association of σ70 and β′ with the rrn Operon A representative rrn operon is shown in black bars, and the orange bands indicate the positions that were probed for σ70 and β′ (RNAP) binding. The numbers represent positions from the major transcription start site, and P1 and P2 represent the major and minor rrn promoters. Relative occupancy (EΔCt) on the y axis represents the enrichment of the DNA immunoprecipitated with antibodies for σ70 or β′ (see Experimental Procedures and Pfaffl, 2001 for details). (A) In rapidly growing cells, σ70 ChIP signal monotonously decreases across the 16S coding region. β′ occupancy is highest at the promoter and then plateaus at a decreased level beyond the promoter. (B) In the presence of rifampicin, both σ70 and β′ are trapped at the promoter. (C) σ70 dissociation was plotted versus time and fit to an exponential decay equation (EΔCt=y0+(max−y0)eoff−kt) to determine the approximate off rate. Assuming an elongation rate of 90 nt/s, we determined a half-life of ∼4 s (t1/2 = ln(2)/koff). The inset presents a corrected plot of the ratio σ70/β versus time, which gave a half-life of ∼7 s. Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Association of σS, σ70, and β′ with rrn and gadA in Stationary Phase (A) σS is present at the rrn promoter, but not in the operon, under stationary-phase growth conditions. β′ occupancy displays some polarity under these conditions. In the presence of rifampicin (lower), EσS and very low levels of Eσ70 are trapped at the promoter. (B) For the stationary-phase-specific gene gadA, EσS binds the promoter (P) robustly and is detected at 550 bp, but σS dissociates completely by 1290 bp, whereas high levels of β′ are found downstream. In the presence of rifampicin, holoenzyme is immobilized at the promoter. Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Association of σ32, σ70, and β′ with the Transcribed Regions of rrn and htpG The data at specific times after a temperature shift from 37° to 45°C are shown. (A) Eσ at the rrn operon. At 5 min after heat shock, Eσ70 and RNAP levels decrease, whereas the levels of Eσ32 increase. By 10 min, Eσ70 levels begin to recover whereas σ32 occupancy decreases. By 20 min, the levels of Eσ70 approach exponential-phase growth values. (B) Eσ at the htpG gene. At 0 min, a measurable amount of Eσ32 is at the promoter. 5 min after heat shock, Eσ32 and RNAP are bound at the htpG operon. The occupancy of σ32 declines by 10 min, but RNAP (β′) levels within the gene remain high. The levels decline further by 20 min. σ70 levels remain at background levels at this gene. Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Association of σ54 and β′ with rrn and with the glnAP2 Promoter σ54 is not detected at the rrn operons in noninducing conditions. The levels of Eσ54 at the nitrogen assimilation gene glnA in log phase in the presence and absence of rifampicin are shown. Both σ54 and β′ are present at the glnA promoter, but not in the gene. Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Conserved Domain Structure of E. coli σ Factors and a Model for σ-Release (A) The conserved domains of σ (regions 1, 2, 3, and 4) are indicated by colored boxes. σ70, σS, and σ32 are all part of the σ70 family with homologous amino acid sequences. σ54 displays poor sequence similarity to the other σ factors. σ70-type of σ factors likely utilize conserved interfaces to associate with the core RNAP as well as with the promoter (regions 2.4 and 4.2). (B) Model of stochastic σ release. The structural model of holoenzyme (holo) and the elongating core (core) is from Darst and coworkers (Murakami et al., 2002). The domains of σ are colored as in (A) (only the crystallized portion of region 1 is in orange), and core RNAP is in gray. In the first step of σ release, the linker between σ region-3 and -4 (blue) is displaced from the RNA exit tunnel in the core by the nascent RNA transcript. Next, interactions between σ region 4 (red) and the β-flap are disrupted by the 15–16 nt transcript (>11 nt). The remaining interactions primarily occur between σ regions 2 plus region 3 (green) and RNAP, and σ factors dissociate from this complex in a stochastic manner. Molecular Cell 2005 20, 357-366DOI: (10.1016/j.molcel.2005.10.011) Copyright © 2005 Elsevier Inc. Terms and Conditions